建立MRC-5细胞三维立体培养模型的探索

发布时间：2018-01-10 20:30

本文关键词：建立MRC-5细胞三维立体培养模型的探索　出处：《中国疾病预防控制中心》2017年硕士论文　论文类型：学位论文

【摘要】：人胚肺成纤维细胞(human embryonic lung fibroblast,MRC-5)来源于 14 周人胚胎的肺组织,可在体外持续培养40多代,并且保持正常的二倍体。MRC-5细胞对许多病毒都具有易感性,如:巨细胞病毒、单纯疱疹病毒、肠道病毒71型等。当前研究MRC-5细胞主要是基于传统的二维细胞培养技术,即细胞在塑料培养瓶或培养皿的平面上单层生长。然而,细胞在体内生长的状态则是三维立体环境,且具有复杂和动态的特性。二维细胞培养由于其自身的局限性,不能充分反映真实组织细胞的生理活性。三维细胞培养弥补了二维细胞培养的缺陷与不足,三维培养模型可在更接近生理环境下研究细胞生物学、细胞生理学及细胞病理学等。本论文实验分别以鼠尾I型胶原和聚苯乙烯(polystyrene,PS)为支架构建两种不同的MRC-5细胞三维培养模型,探索在这两种模型中常规培养液、血小板衍生生长因子培养液(platelet-derived growth factor,PDGF)和支气管上皮细胞生长培养液(bronchial epithelial cell growth medium,BEGM)对 MRC-5 细胞生长状态的影响,进一步比较这两种培养模型中MRC-5细胞的生长状态的差异,初步观察在这两种培养模型中MRC-5细胞感染人巨细胞病毒(human cytomegalovirus,HCMV)的病变状况。实验结果显示,常规培养液、PDGF培养液和BEGM培养液均能使细胞在这两种培养模型中相互交错生长,形成多层立体的三维网状结构。其中在三维胶原-MRC-5细胞培养模型中,PDGF培养液有利于早期促进细胞呈树突状伸展,常规培养液和BEGM培养液则更有利于MRC-5细胞在较长时间内保持三维立体的网状结构。常规培养液可促进细胞在胶原表面聚集,PDGF培养液和BEGM培养液则有利于细胞以单个细胞方式进行迁移。在三维PS-MRC-5细胞培养模型中,与常规培养液和PDGF培养液相比,BEGM培养液中生长的MRC-5细胞能够形成更加明显的三维立体网状或“鸟巢状”结构,且能长时间保持。与三维胶原基质内生长的MRC-5细胞相比较,在三维PS支架中生长的MRC-5细胞扩展延伸的速度相对较快,但维持细胞三维立体结构的时间相对较短。在上述两种培养模型内生长的MRC-5细胞均能感染HCMV病毒,并且可呈现明显的细胞病变效应(cytopathic effect,CPE),说明MRC-5细胞没有因为改变培养体系而改变其对HCMV病毒的易感性。与三维胶原基质内MRC-5细胞的病变相比较,三维PS支架内的MRC-5细胞出现细胞病变的时间相对较早,病变持续时间相对较短,病变程度相对较高。通过我们的探索研究,利用鼠尾Ⅰ型胶原和聚苯乙烯材料成功地构建了两种不同类型的MRC-5细胞三维培养模型,并且在两种培养模型中生长的MRC-5细胞对HCMV病毒的易感性没有发生改变。这为进一步优化MRC-5细胞三维培养模型和利用MRC-5细胞三维培养模型研究病毒感染、增殖及抗病毒药物等提供实验依据。
[Abstract]:Human embryonic lung fibroblast cells (human embryonic lung fibroblast, MRC-5) from the 14 week human fetal lung tissue, in vitro cultured for more than 40 generations continuously, and maintain the normal diploid.MRC-5 cells have susceptibility to many viruses such as cytomegalovirus, Dan Chunpao measles virus, enterovirus 71. Current research MRC-5 cell culture technology is mainly based on the traditional two-dimensional cell, in which cells grown in monolayer culture or plastic bottle dish on the plane. However, the growth of cells in the body state is the three-dimensional environment, and has the characteristics of complex and dynamic. The two-dimensional cell culture because of its own limitations, can not fully reflect the true physiological activity tissue cells. Three dimensional cell culture to make up for deficiencies in the two-dimensional cell culture and the lack of three-dimensional culture model can be closer to the physiological environment of cell biology, cell physiology And cell pathology. The experiment respectively with rat tail collagen type I and polystyrene (polystyrene, PS) to construct two MRC-5 three-dimensional cell culture model for different support, explore the common medium in the two model, platelet-derived growth factor (platelet-derived growth medium factor, PDGF) and bronchial epithelial cells the growth medium (bronchial epithelial cell growth medium, BEGM) growth effect on MRC-5 cells, further comparison of the two kinds of culture model of MRC-5 cells in the growing state of difference, preliminary observation on these two kinds of cultured human cytomegalovirus MRC-5 cell infection model (human cytomegalovirus, HCMV). The experimental results of lesions show that the conventional culture medium, PDGF medium and BEGM medium can make the cell culture model in the two mutually staggered growth, formation of multi layer three-dimensional mesh structure Which in three-dimensional collagen -MRC-5 cell culture model, PDGF culture medium is conducive to the early promotion of dendritic cells were stretched, common culture medium and BEGM culture medium is more conducive to MRC-5 cells maintain a three-dimensional reticular structure in a long time. The conventional culture medium could promote the accumulation of cells in collagen surface, PDGF medium and BEGM medium is conducive to the cells in a single cell by way of transfer. In the 3D PS-MRC-5 cell culture model, compared with the conventional culture medium and PDGF culture medium, BEGM culture of MRC-5 cells grown in liquid to form a three-dimensional network more obvious or "bird nest" structure, and can be kept for a long time. With the growth of three-dimensional collagen matrix the growth in MRC-5 cells compared to MRC-5 cells in three-dimensional PS scaffold extended relatively fast speed extension, but maintaining cells in three-dimensional structure in a relatively short time. The two culture model the growth of MRC-5 cells could be infected with the HCMV virus, and showed obvious cytopathic effects (cytopathic effect, CPE), indicating that MRC-5 cells did not change because the culture system and change its susceptibility to HCMV virus. MRC-5 cells and three-dimensional collagen matrix in the lesions compared to 3D PS stent. MRC-5 cytopathic time relatively early, in the relatively short duration of disease, a relatively high degree. Through our research, the successful construction of the two kinds of different types of MRC-5 three-dimensional cell culture model using rat tail collagen and polystyrene material, and there is no change in the growth of the susceptibility of two kinds of culture MRC-5 cell model of HCMV virus. This in order to further optimize the three-dimensional culture model of MRC-5 cells and MRC-5 cells using three-dimensional culture model to study the virus infection and proliferation It provides experimental basis for antiviral drugs and so on.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R329.2

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